倒刺两极虫的宿主新记录及其分子系统学研究 向瑶 张金叶 赵元 NEW HOST RECORD AND MOLECULAR PHYLOGENY OF MYXIDIUM SPINIBARBA CHEN ET AL., 2020 XIANG Yao, ZHANG Jin-Ye, ZHAO Yuan-Jun 在线阅读 View online: https://doi.org/10.7541/2021.-0000-00

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2021 年 11 月 ACTA HYDROBIOLOGICA SINICA Nov., 2021 doi: 10.7541/2021.-0000-00

倒刺鲃两极虫的宿主新记录及其分子系统学研究

向 瑶 张金叶 赵元莙 (重庆师范大学生命科学学院, 动物生物学重庆市重点实验室, 重庆 401331)

摘要: 为弄清倒刺鲃两极虫(Myxidium spinibarba)的宿主多样性和胭脂鱼(Myxocyprinus asiaticus)寄生黏孢子 虫的种类组成, 研究基于形态和分子数据, 比较分析了寄生于不同宿主的倒刺鲃两极虫的形态学和形态计量 学特征及分子系统发育关系。结果显示: 寄生于胭脂鱼和中华倒刺鲃的倒刺鲃两极虫株系在形态学和形态计 量学上未出现显著性差异, 18S rDNA序列相似度为99.9%—100.0%, 遗传距离为0.000—0.001, 符合种内变异; 寄生于不同宿主倒刺鲃两极虫的株系在系统发育树中嵌合聚支, 且寄生于胭脂鱼的倒刺鲃两极虫株系先分 化。以上结果表明: 研究中两株系与倒刺鲃两极虫为同一物种, 但在分子水平已经出现分化; 这是首次在胭脂 鱼中检获到黏孢子虫, 胭脂鱼是倒刺鲃两极虫的新宿主。

关键词: 倒刺鲃两极虫; 胭脂鱼; 宿主新记录; 分子系统学; 18S rDNA 中图分类号: S941.51 文献标识码: A 文章编号: 1000-3207(2021)06-0001-08

黏孢子虫是一类个体微小结构简单的后生动 囊或泌尿系统, 极少数可营组织寄生[2, 17], 个别组织 物寄生虫, 主要寄生于鱼类, 有些种类能引起鱼类 寄生种类甚至能导致寄生部位出现空泡影响宿主 宿主的病害[1—3], 严重的甚至会导致死亡[4—7]。其中 发育和繁殖[18]。 两极虫科Myxidiidae Thélohan, 1892黏孢子虫与大 胭脂鱼Myxocyprinus asiaticus Bleeker, 1864, 多数黏孢子虫类群不同, 具有最广泛的宿主类群, 隶属鲤形目Cypriniformes胭脂鱼科Catostomidae胭 该科的种类不仅能寄生于变温动物(海、淡水鱼 脂鱼属Myxocyprinus, 是胭脂鱼科唯一分布在我国 类、两栖类、爬行类)[8—12], 还能寄生于恒温动物 的特有种[19, 20], 现已在我国驯化为养殖鱼类, 成为 (鸟类、哺乳类)[13, 14]。有些物种还能广泛寄生于多 我国长江流域重要经济鱼类之一[21, 22]。胭脂鱼是 种不同的宿主, 如李氏肠黏虫Enteromyxum leei 杂食性的底栖鱼类, 在人工养殖过程中常与其他不 Diamant, Lom and Dykova, 1994的宿主已经超过 同食性和不同水层栖息的鱼类混养以充分利用不 [23] 40种[15]。两极虫属Myxidium Butschli, 1882隶属黏 同水层的资源 。但混养易增加胭脂鱼与其他不 体门Myxozoa、黏孢子虫纲Myxosporea、双壳目 同鱼类的密切接触机会, 加大寄生虫或其他病原传 [24] Bivalvulida、两极虫科Myxidiidae[16], 是两极虫科 播的风险, 进而增高传染性造成经济损失 。因此 中种类最多的属, 迄今已记录的种类超过230种[9]。 开展对胭脂鱼的寄生虫及其病害的研究也是水产 两极虫属种类具如下特征: 2个孢子壳瓣, 孢子呈纺 养殖中鱼病防治的需要。 锤形、梨形或椭圆形, 末端稍尖; 壳瓣表面光滑或 倒刺鲃两极虫Myxidium spinibarba Chen et al., 具条纹, 孢子缝线直; 孢子的两极端各具一个梨形 2020最初从中华倒刺鲃Spinibarbus sinensis (Bleeker, 或球形的极囊且极囊向两端开口预出版, 细长的极丝在极 1871) 的胆囊中检获[25]。本研究检获的倒刺鲃两极 囊内横向盘曲; 孢子质位于两极囊之间。两极虫为 虫分离自胭脂鱼胆囊, 在对其重描述的基础上, 结 典型的腔寄生类群, 绝大多数种类寄生于宿主的胆 合其形态学与18S rDNA分子数据, 比较分析寄生

收稿日期: 2020-08-18; 修订日期: 2021-02-06 基金项目: 国家自然科学基金 (31970409和31672280); 重庆市自然科学基金 (cstc2014jcyjA00006); 重庆市教委科学技术研究项目 (KJ1400515)资助 [Supported by the National Natural Science Foundation of China (31970409 and 31672280); Project of Chongqing Science & Technology Commission (cstc2014jcyjA00006); Science & Technology Research Foundation of Education Committee of Chongqing (KJ1400515)] 作者简介: 向瑶(1996—), 女, 硕士研究生; 研究方向为鱼类寄生虫学。E-mail: [email protected] 通信作者: 赵元莙, E-mail: [email protected] 2 水 生 生 物 学 报 45 卷

于不同宿主的倒刺鲃两极虫及其他相似种之间的 关研究所用序列[25]及本研究所获的2条序列进行系 分子系统学关系, 旨在为寄生胭脂鱼的黏孢子虫种 统发育分析。另以Tetracapsuloides bryosalmonae 类组成及两极虫的宿主多样性积累基础数据。 (KF731712)和Buddenbrockia plumatellae(KF731691) 为外群, 利用Mr Bayes 3.2.6[30]选用最优拟合模型 1 材料与方法 (GTR)运行1000000代构建Bayes(BI)树, 利用在线 1.1 黏孢子虫样本采集和鉴定 软件CIPRES Science Gateway V.3.3(http://www.phylo. 宿主鱼于2019年10月11日从重庆北碚区某养 org/)选用RAxML-HPC2 XSEDE(8.2.12)模型构建 殖基地(该养殖基地由8个8.35 m×5.66 m的养殖池 ML(Maximum likelihood)树。最后用FigTree 相连, 且池水互通; 多种鱼类以不同年龄、不同规 v1.4和Adobe Photoshop CC软件编辑注释系统树。 [31] 格同池或者单独轮养轮放)捕获。共获26尾无明显 将相关18S rDNA序列用MEGA 6.0 设置执行 病症的胭脂鱼带回实验室解剖并进行粘孢子虫检 K2P模型多序列比对计算出遗传距离, 利用在线序 测, 其中4尾鱼的胆囊检获到黏孢子虫(感染率为 列比对工具(http://www.ebi.ac.uk/Tools/psa/)计算得 15.4%)。黏孢子虫收集、处理、测量和鉴定方法 出两两序列相似度, 结合MEGA和Bioedit软件完成 参照文献[26]处理。 变异位点分析。 1.2 DNA提取、PCR扩增和测序 1.4 主成分分析 从上述同一批次不同胭脂鱼个体的胆囊内分 使用PAST 3软件进行主成分分析(Principal 离获得游离黏孢子虫, 加入95%的乙醇溶液置于1.5 mL Component Analysis, PCA), 对不同宿主寄生的倒刺 离心管保存。从离心管底部吸取10 μL黏孢子虫液 鲃两极虫株系的孢子长、孢子宽、极囊长、极囊 [32] 体, 用超纯水清洗2—3次, 离心(2000 ×g)沉淀后用 宽和极囊间距5项形态学特征进行形态计量学比较 。 于DNA提取。基因组DNA的提取严格参照DNeasy 利用变异协方差矩阵生成置信度为95%的散射图。 试剂盒 Blood & Tissue (QIAGEN, Hilden, Germany) 2 结果 说明书执行, 将提取的基因组DNA置于-20℃保 存、备用。黏孢子虫18S rDNA序列通过PCR技术 2.1 倒刺鲃两极虫的形态学重描述 利用引物18e: 5′-CTGGTTGATCCTGCC-3′[27]和 取宿主鱼的适量胆汁制片镜检, 未见营养体和 18R: 5′-CTACGGAAACCTTGTTACG-3′[28]扩增。 未成熟孢子, 但可见大量成熟孢子。成熟孢子壳面 反应体系和反应条件参照文献[29]。取3 μL扩增产 呈梭形, 两端稍尖。孢子缝面呈卵圆形, 缝脊平直, 物用于1%琼脂糖凝胶进行电泳检测, 目的产物经 孢子壳瓣上有7—10条与缝脊平行的条纹(图 1)。 胶回收试剂盒(OMEGA, 美国)回收并送到上海英 孢子长为(12.59±0.56) μm (11.55—13.36 μm), 孢子 潍捷基贸易有限公司测序。 宽为(5.92±0.40) μm (5.11—6.86 μm)。两个极囊呈 1.3 系统发育分析 梨形位于孢子两端, 大小相等, 其开口倾向孢子两 将本研究获得的序列, 在GenBank中用BLAST 端。极囊长为(3.48±0.36) μm (2.63—4.22 μm), 极 进行同源序列检索比对。根据比对结果, 选取12条 囊宽为(2.83±0.26) μm (2.29—3.21 μm)。两极囊间 高同源性序列(序列相似度高于89%), 24条先前相 隔(3.66±0.29) μm (3.12—4.20 μm), 极丝盘曲 a 预出版b c

图 1 寄生于胭脂鱼的倒刺鲃两极虫孢子显微图片 Fig. 1 Photomicrographs of Myxidium spinibarba from Myxocyprinus asiaticus a. 孢子壳面观(箭头示孢子尖端); b. 孢子斜缝面观(箭头示孢子缝线); c. 孢子壳面观(箭头示孢子壳面条纹); 比例尺=10 μm a. Spore from valvular view (arrowhead showing protrusive poles); b. Spore from slant sutural view (arrowhead showing sutural line); c. Spore from valvular view (arrowhead showing spore striations). Scale bar=10 μm 6 期 向 瑶等: 倒刺鲃两极虫的宿主新记录及其分子系统学研究 3

4—5圈(表 1)。 亚支再形成了姐妹支。 2.2 18S rDNA分子特征和系统发育分析 2.3 基于寄生不同宿主的倒刺鲃两极虫形态学数 本研究从不同个体的胭脂鱼样本中获得2条 据的主成分分析 18S rDNA序列, 长度分别为1785(MT775466)和 主成分分析结果显示本研究两株系(MT775466 1913 nt (MT775467)。比对结果显示, 所获两条18S 和MT775467)与倒刺鲃两极虫(MH766654; 该株系 rDNA序列之间相似度为100.00%, 在可比对长度 原始数据来自本实验室)在5项形态特征结合生成 (1785 nt)上无变异位点。两条序列在NCBI中 的椭圆散点图上均有重合, 其中●株系(MT775466) BLAST分析显示: 与寄生于中华倒刺鲃的倒刺鲃两 散点图基本覆盖*株系(MH766654)散点图, ▲株系 极虫(MH766654)的相似度最高, 为99.9%—100.0%, (MT775467)的散点图仅与前两者的散点图部分重 MT775467与Myxidium spinibarba (MH766654)在应 叠(图 3)。 可比对长度范围内(1913 nt)仅有1个变异位点; 其次 3 讨论 与Myxidium sp.(MK913426)和Zschokkella sp.(KM401441)具有较高相似度(表 2)。 本研究两株系形态学和生态学特性均显示一 基于18S rDNA序列构建的ML和BI树显示出 致, 应属于同一物种。本研究获得的两极虫, 两端 一致的拓扑结构。两棵系统树均显示: 所选取黏孢 稍尖的梭形成熟孢子、平直的缝脊和壳瓣上的条 子虫序列被分成A、B两个大分支(图 2)。A支是一 纹数、两个大小相等的梨形极囊、极丝圈数及极 混合支系, 包括寄生于爬行动物的两极虫、海水鱼 丝开口方向等形态学特征均与倒刺鲃两极虫的形 类的两极虫和楚克拉虫及淡水鱼类的两极虫和楚 态学特征一致[25], 且两者的感染部位均为胆囊。同 克拉虫; 而B支则完全由寄生海水鱼类的两极虫组 时, 形态学数据的主成分分析结果也显示: 尽管株 成。本研究株系位于A支内, 其中株系(MT775466) 系(MT775467)有略微的差异, 但本研究的两个株系 与倒刺鲃两极虫(MH766654)聚支后, 再与株系 的形态测量数据基本都与倒刺鲃两极虫的形态测 (MT775467)聚支, 形成倒刺鲃两极虫支系; 然后, 与 量数据有大幅重叠(图 3), 仅存在少许差异, 这样的 Zschokkella sp.(KM401441)和Myxidium sp.(MK913426) 形态学量度差异在种内变化范围内[33]。综合形态

表 1 寄生不同宿主的倒刺鲃两极虫的特征比较 Tab. 1 Comparison among Myxidium spinibarba from different hosts 株系(登录号) Strains 倒刺鲃两极虫(MT775466) 倒刺鲃两极虫(MT775467) 倒刺鲃两极虫(MH766654) (Accession No.) Myxidium spinibarba (MT775466) Myxidium spinibarba (MT775467) Myxidium spinibarba (MH766654) 18S rDNA序列长度18S rDNA sequence length (nt) 1785 1913 1949 宿主Host 胭脂鱼Myxocyprinus asiaticus 胭脂鱼Myxocyprinus asiaticus 中华倒刺鲃Spinibarbus sinensis 寄生部位Infection site 胆囊Gall bladder 胆囊Gall bladder 胆囊Gall bladder 孢子长Spore length (μm) 12.6±0.56 (11.6—13.4) 13.0±0.39 (12.3—13.6) 11.8±0.5 (10.6—12.4) 孢子宽Spore width (μm) 5.9±0.40 (5.1—6.9) 5.7±0.45 (5.0—6.3) 6.1±0.5 (5.5—7.2) 极囊长Polar capsule length (μm) 3.5±0.36 (2.6—4.2) 3.5±0.30 (2.7—4.0) 3.6±0.4 (3.0—4.4) 极囊宽Polar capsule width (μm) 2.8±0.26 (2.3—3.2) 2.9±0.22 (2.3—3.4) 3.0±0.2 (2.7—3.2) 极囊间距 Space between polar capsule (μm) 3.7±0.29 (3.1—4.2) 3.9±0.36 (3.2—4.7) 3.6±0.5 (2.8—4.2) 极丝圈数Polar filament number (μm) 4—5 4—5 4—5 [25] 数据来源Reference 本研究预出版Present study 本研究Present study Chen等, 2020 Chen et al., 2020 表 2 黏孢子虫基于18S rDNA序列遗传距离与相似度 Tab. 2 Genetic distances and similarities of myxosporeans based on 18S rDNA sequences

物种名Species GenBank登录号 (GenBank accession No.) 1 2 3 4 5 1. Myxidium spinibarba MT775466 99.9% 100.0% 97.7% 97.4% 2. Myxidium spinibarba MT775467 0.001 99.9% 97.6% 97.4% 3. Myxidium spinibarba MH766654 0.000 0.001 97.7% 97.5% 4. Myxidium sp. MK913426 0.021 0.022 0.021 97.2% 5. Zschokkella sp. KM401441 0.022 0.023 0.022 0.028 4 水 生 生 物 学 报 45 卷

1.00/98 Zschokkella candia MG980597 1.00/100 Zschokkella sp. DQ333435 1.00/70 Zschokkella icterica DQ333434 0.68/60 Zschokkella sp. MN211361 1.00/72 Zschokkella auratis KC849425 1.00/100 Zschokkella trachini KU588385 18S rDNA Zschokkella sp. DQ377704 1.00/99 Zschokkella soleae JX271832 BI/ML Zschokkella sp. KY475591 0.64/33 1.00/100 0.07 1.00/97 Zschokkella sp. DQ118776 0.98/81 Zschokkella parasiluri DQ377689 0.69/42 Myxidium cuneiforme DQ377709 Myxidium shedkoae MG748712 0.97/62 Zschokkella nova DQ377690 1.00/81 目 科 1.00/100 Myxidium truttae AF201374 宿 主 / 形态

0.33/65 Myxidium spinibarba MT775466 Myxocyprinus asiaticus /Catostomidae 梭

0.94/83 Myxidium spinibarba MH766654 Spinibarbus sinensis Cypriniformes/Cyprininae 形 1.00/76 1.00/97 Myxidium spinibarba MT775467 Myxocyprinus asiaticus Cypriniformes/Catostomidae 孢 Zschokkella sp. KM401441 rohita Cypriniformes/ 0.99/98 子 0.99/84 Myxidium sp. MK913426 Cranoglanis bouderius Siluriformes/Cranoglanididae 1.00/88 Myxidium sp. KP030766 Labeo horie Cypriniformes/Labeoninae Myxidium amazonense KT625442 1.00/100 Myxidium hardella AY688957 A 1.00/100 Myxidium chelonarum DQ377694 1.00/86 Myxidium turturibus KX611144 1.00/95 Myxidium peruviensis KY996746 Myxidium coryphaenoideum DQ377697 0.98/83 1.00/100 Myxidium baueri JX467674 1.00/79 Myxidium laticurvum JN033230 1.00/75 Myxidium lapipiscis MG562499 Myxidium scomberomori KF179052 0.54/48 1.00/100 Myxidium incurvatum DQ377708 B 1.00/100 Myxidium milleri KF179053 0.91/73 Myxidium maxi KF179054 Myxidium bergense DQ377702 0.97/81 Myxidium gadi DQ377711 1.00/100 Tetracapsuloides bryosalmonae KF731712 Buddenbrockia plumatellae KF731691 图 2 基于18S rDNA序列构建的BI/ML系统发育树 Fig. 2 Phylogenetic tree generated by BI/ML based on 18S rDNA sequences 节点表示支持率; 物种名后为GenBank登录号 The node represents support value; GenBank accession number is after the species names

3.00 株系与中华倒刺鲃株系之间仅有1个变异位点, 遗 2.25 传距离和相似度分别为0.000—0.001和99.9%— 1.50 100.0%, 符合已有研究给出的黏孢子虫变异位点不 0.75 超过10个, 遗传距离在0.000—0.007, 相似度在 −3.00 −2.25 −1.50 −0.75 0.75 1.50 2.25 3.00 [36—39] −0.75 98.6%—100%属于种内水平的判别标准 。分 Component 2 −1.50 子特征分析也表明本研究所获寄生胭脂鱼的两极 −2.25 虫株系(MT77546和MT775467)与寄生中华倒刺鲃 −3.00 Component 1 的倒刺鲃两极虫(MH766654)为同一物种。 系统发育分析显示: 寄生不同宿主的倒刺鲃两 图 寄生不同宿主倒刺鲃两极虫的孢子形态差异主成分分析 3 极虫株系嵌合聚枝, 寄生胭脂鱼的倒刺鲃两极虫株 Fig. 3 Principal component analysis on morphological differences 系(MT775467)先分化, 并与前述的主成分分析和 of Myxidium spinibarba from different hosts 特征分析结果表现一致。同种两极虫可 * 倒刺鲃两极虫(MH766654); ● 倒刺鲃两极虫(MT775466); ▲ 18S rDNA 倒刺鲃两极虫(MT775467) 在亲缘关系较远的鱼类宿主寄生, 这与已有的关于 [33, 40] * Myxidium spinibarba (MH766654); ● Myxidium spinibarba 弧形虫和肠黏虫的研究有着明显的一致性 。 (MT775466); ▲ Myxidium spinibarba (MT775467) 在本研究所显示的倒刺鲃两极虫姐妹支中, 寄生于 预出版露斯塔野鲮Labeo rohita Hamilton, 1822(鲤形目)的 学与主成分分析结果, 本研究获得的两极虫与倒刺 Zschokkella sp.(KM401441)与寄生于长臀Cran- 鲃两极虫当属同种。 oglanis bouderius Richardson, 1846(鲇形目)的Myx- 鉴于黏孢子虫结构简单, 学者们在以往的研究 idium sp.(MK913426)形成高支持率(0.99/84)的系统 中认为在对黏孢子虫进行物种鉴定和描述时, 应当 发生关系也得到旁证: 即尽管Zschokkella sp.(KM 综合考虑该黏孢子虫的形态学特征、生态学特征 401441)与Myxidium sp.(MK913426)为不同种类且 [34-35] (宿主、寄生部位、地理分布)和分子特征 。基 宿主亲缘关系较远, 但显示形态学特征较为相似, 于此, 尽管本研究株系的宿主与原始描述宿主不同, 都享有梭形的孢子并具有梨形极囊(图 2)。两极虫 但其18S rDNA序列比对结果显示寄生胭脂鱼的两 的系统发生关系与孢子形态、腔寄生的寄生部位 6 期 向 瑶等: 倒刺鲃两极虫的宿主新记录及其分子系统学研究 5

趋向性等可能更相关。在通常情况下, 内寄生虫的 tion of Enteromyxum scophthalmi gen. nov. sp. nov. 进化在很大程度上受其与宿主相互作用的影响, 基 (Myxozoa), an intestinal parasite of turbot (Scophthalmus 于黏孢子虫SSU rDNA的大数据集系统发育分析结 maximus L.) using morphological and ribosomal RNA se- quence data [J]. Parasitology, 2002, 124(4): 369-379. 果认为组织寄生物种是由类似祖先型的腔寄生黏 [8] Whipps C, Zhao Y J. Synopsis of the species of the genus [41, 42] 孢子虫进化而来 。寄生鱼类的碘泡虫类黏孢 Sphaeromyxa Thélohan, 1892 (Myxosporea: Bivalvulida: [42] 子虫多为组织寄生, 是黏孢子虫中进化较晚的类群 。 Variisporina: Sphaeromyxidae) [J]. Systematic Parasito- 绝大多数碘泡虫类的黏孢子虫寄生于淡水鱼且具 logy, 2015, 92(2): 81-99. 有明显的宿主特异性乃至寄生部位特异性, 往往只 [9] Eiras J C, Saraiva A, Santos M J, et al. Synopsis of the 感染一种宿主甚至只感染一种宿主的某一特定器 species of Myxidium Bütschli, 1882 (Myxozoa: Myxosporea: Bivalvulida) [J]. Systematics Parasitology, 官或组织[35]。而腔寄生的两极虫类是相对组织寄 2011, 80(2): 81-116. 生的碘泡虫类较为祖先的类群 进化过程中分化形 , [10] Hartigan A, Fiala I, Dyková I, et al. New species of 成海水支系和淡水支系, 其变异程度要比碘泡虫等 Myxosporea from frogs and resurrection of the genus 组织寄生类群更大, 更易于对环境及宿主的变化形 Cystodiscus Lutz, 1889 for species with myxospores in 成适应[41, -42]。此外, 先前基于鱼体混养、受感染鱼 gallbladders of amphibians [J]. Parasitology, 2012, 宿主的生活水体、经口饲喂和肛门插管等途径对 139(4): 478-496. [11] Heiniger H, Adlard RD. Relatedness of novel species of 李氏肠黏虫传播实验研究表明, 李氏肠黏虫可感染 Myxidium Bütschli, 1882, Zschokkella Auerbach, 1910 多种不同且亲缘关系较远的鱼类宿主[43, 44] 也说明 , and Ellipsomyxa Køie, 2003 (Myxosporea: Bivalvulida) 腔寄生的两极虫类黏孢子虫对宿主具有更广泛的 from the gall bladders of marine fishes (Teleostei) from 适应性。鉴于此, 推测本研究中倒刺鲃两极虫也可 Australian waters [J]. Systematic Parasitology, 2014, 能具有多宿主特征, 能寄生多种不同宿主, 但这还 87(1): 47-72. 需要更多研究予以证实。 [12] Espinoza L L, Mertins O, Gama G S, et al. A new Myx- idium species (Myxozoa: Myxosporea) infecting the gall- 综上, 本研究首次在胭脂鱼体内检获黏孢子虫, bladder of the turtle Podocnemis unifilis (Testudines: 胭脂鱼是倒刺鲃两极虫的新宿主; 寄生胭脂鱼的倒 Podocnemididae) from Peruvian Amazon [J]. Acta Trop- 刺鲃两极虫株系(MT775467)先分化。本研究也为 ica, 2017(172): 75-79. 寄生胭脂鱼的黏孢子虫种类组成积累了新的数据。 [13] Bartholomew J L, Atkinson S D, Hallett S L, et al. Myxo- zoan parasitism in waterfowl [J]. International Journal 参考文献: for Parasitology, 2008, 38(10): 1199-1207. [1] Kent M L, Andree K B, Bartholomew J L, et al. Recent [14] Szekely C, Cech G, Atkinson S D, et al. A novel myxozo- advances in our knowledge of the Myxozoa [J]. Journal an parasite of terrestrial mammals: description of Sor- of Eukaryotic Microbiology, 2001, 48(4): 395-413. icimyxum minuti sp n. (Myxosporea) in pygmy shrew [2] Lom J, Dyková I. Myxozoan genera: definition and notes Sorex minutus from Hungary [J]. Folia Parasitologica, on , life-cycle terminology and pathogenic spe- 2015, 62(45): 1-5. cies [J]. Folia Parasitologica, 2006, 53(1): 1-36. [15] Diamant A, Ram S, Paperna I. Experimental transmission [3] Molnár K. Site preference of myxozoans in the kidneys of of Enteromyxum leei to freshwater fish [J]. Diseases of Hungarian fishes [J]. Diseases of Aquatic Organisms, Aquatic Organisms, 2006, 72(2): 171-178. 2007, 78(1): 45-53. [16] Canning E U, Okamura B. Biodiversity and evolution of [4] Kazuo K. Intestinal giant-cystic disease affecting the carp, the Myxozoa [J]. Advances in Parasitology, 2003, 56(2): caused by Thellohanellus sp [J]. Fish Pathology, 1980, 43-131. 14(3): 145-146. [17] Fariya N, Kaur H, Abidi R. Myxidium tictoi n. sp. a [5] Zhai Y H, Gu Z M, Guo Q X, et预出版 al. New type of patho- myxozoan parasite infecting kidney of fresh water barb genicity of Thelohanellus kitauei Egusa & Nakajima, Puntius ticto (Hamilton, 1822) from river Gomti, Luc- 1981 infecting the skin of common carp Cyprinus carpio know (U. P) [J]. Journal of Parasitic Diseases, 2019, L [J]. Parasitology International, 2016, 65(1): 78-82. 44(4): 126-130. [6] El-Matbouli M, Hoffmann R W, Schoel H, et al. Whirl- [18] Mohammed A J, Reda H. Ovarian abnormality in a patho- ing disease: host specificity and interaction between the logical case caused by Myxidium sp. (Myxozoa, actinosporean stage of Myxobolus cerebralis and rainbow Myxosporea) in onespot snapper fish Lutjanus mono- trout Oncorhynchus mykiss [J]. Diseases of Aquatic Or- stigma (Teleostei, Lutjanidae) from the Red Sea [J]. Acta ganisms, 1999, 35(1): 1-12. Parasitologica, 2010, 55(1): 1-7. [7] Palenzuela O, Redondo M J, Álvarez-Pellitero P. Descrip- [19] Luo Y L, Wu X W. Anatomical feature of Myxocyprinus 6 水 生 生 物 学 报 45 卷

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NEW HOST RECORD AND MOLECULAR PHYLOGENY OF MYXIDIUM SPINIBARBA CHEN ET AL., 2020

XIANG Yao, ZHANG Jin-Ye and ZHAO Yuan-Jun (Chongqing Key Laboratory of Biology, College of Life Sciences, Chongqing Normal University, Chongqing 401331, China)

Abstract: Myxosporeans are microscopic and simple in morphology, mainly parasitize in fish. Some species can cause host disease even lead to death. Among them, the species of the family Myxidiidae Thélohan, 1892 exhibit the broadest range of hosts among all myxosporeans. They are typically coelozoic (rarely histozoic) parasites of marine and freshwa- ter fishes (poikilothermal ), and a few species are found in another poikilothermal animals (amphibians and rep- tiles) and homothermal animals (birds and mammals). The genus Myxidium Bütschli, 1882 with 230 described species, currently includes the most known species in the family Myxidiidae. The members of the genus Myxidium exhibit the following characteristics: their myxospores are spindle-shaped, straight, crescent, or even sigmoid with somewhat poin- ted ends; two pyriform polar capsules are located at both ends of the myxospores; the shell valves are smooth or stri- ated; and the suture line bisects the myxospores. Myxidium spinibarba Chen et al., 2020 was first collected and de- scribed from the gall bladder of Spinibarbus sinensis Bleeker, 1871. Present study reported M. spinibarba again from Myxocyprinus asiaticus Bleeker, 1864. Myxocyprinus asiaticus belongs to the family Catostomidaeis, in which only one species has been reported from China. Myxocyprinus. asiaticus is a kind of omnivorous benthic fish with important or- namental and edible value. To improve higher economic benefits, M. asiaticus and other fishes with different diets and habitat preferences are often mixed feeding. However, polyculture creates conditions for material exchanges between M. asiaticus and other fishes, and, it also enhances the transmission of parasites or pathogens. To study the host diversity for M. spinibarba and the species composition of myxosporean from M. asiaticus, we analyzed the morphometry characteristics and phylogenetic relationships of M. spinibarba from different hosts based on the morphological and molecular data. Myxosporeans were isolated and photographed from the gallblad- der of different M. asiaticus in the same batch. Genomic DNA were extracted and 18S rDNA was amplified and sequenced. Myxospores were fusiform in frontal view, (12.6±0.6) μm (11.6—13.4 μm) long, (5.9±0.4) μm (5.1—6.9 μm) wide, and possessed distinctly longitudinal ridges. The two polar capsules were pear-shaped at the ends of the spore, with a length in (3.48±0.36)预出版 μm (2.63—4.22 μm) and a width of (2.83±0.26) μm (2.29—3.21 μm) with 4—5 turns polar filaments. The distance between two polar capsules was (3.66±0.29) μm (3.12—4.20 μm), and their openings were inclined towards the spore ends. These two sequences have the highest similarity with M. spinibarba (MH766654) parasitizing in S. sinensis (99.9%—100.0%). They shared 97.4%—97.7% identity with Zschokkella sp. (KM401441) from Labeo rohita Hamilton, 1822 and Myxidium sp. (MK913426) from Cranoglanis bouderius Richardson, 1846, respectively. Phylogenetic trees of the selected sequences were divided into branch A and B. Clade A was composed Myxidium and Zschokkella species from marine and freshwater fishes or amphibians. All species in clade B were collected from marine fish. Two strains in this study were loc- ated in branch A, where the strain (MT775466) and M. spinibarba (MH766654) were first clustered and then 8 水 生 生 物 学 报 45 卷 grouped with the strain (MT775467). The results of PCA showed that the two strains of this study (MT775466 and MT775467) and M. spinibarba (MH766654) overlapped on the scatter plot. The similarities and genetic dis- tances of 18S rDNA sequences for three strains were 99.9%—100.0% and 0.000—0.001, respectively. Therefore, we conclude that these three strains from different hosts are the same species as M. spinibarba, with diverged mo- lecular level, and that this is the first report of myxosporean in M. asiaticus.

Key words: Myxidium spinibarba; Myxocyprinus asiaticus; New host record; Molecular phylogeny; 18S rDNA

预出版